An outbreak of Fusarium head blight occurred in southern NSW in 2000 but was not widespread, being localised around regions where much higher rainfall than average occurred during anthesis in November. The causal fungi included F. graminearum, F. culmorum, F cerealis (= F. crookwellense), F. pseudograminearum and F. avenaceum. The presence of F graminearum in southern NSW was outside the range documented so far in Australia. Sequence analysis revealed that all deoxynivalenol (DON) producing isolates representative of the global lineages of F graminearum displayed characteristic deletions in a region in the upstream sequences of the Tri5 gene compared to the nivalenol (NIV) chemotype. The distinct length polymorphism in this region between the DON and NIV chemotypes forms the basis of a PCR assay in this study to distinguish between them. Six F graminearum isolates from southern NSW and 20 overseas isolates were analysed using this technique. Two of the isolates from southern NSW were of the DON chemotype while the other four were of the NIV chemotype. Further research is required to establish the relative distribution of DON and NIV chemotypes in the NSW and Australian grain-belt.